This invention relates to polymer chemistry and, more particularly, to a polyacrylamide gel electrophoresis column and a method of making the same. A major objective of the invention is to produce a column with a gel matrix that is void-free and that withstands the stress of high-voltage electrophoresis without rupture or displacement.
Polyacrylamide gel electrophoresis (PAGE) is a prominent methodology for analyzing protein mixtures. In a typical PAGE procedure, a sample protein mixture is introduced at one end of a capillary column, which can be a fused silica tube filled with a polyacrylamide gel matrix. The gel matrix has a lattice structure with pores that act as a molecular sieve, allowing small particles to pass easily, but imparting frictional drag to larger particles. Charged biomolecules migrate through the gel within the electric field at speeds inversely related to their molecular weight. The differential speeds cause mixture components to separate into bands that can be detected as they pass a detector to provide an analysis. Where it is desirable to isolate a protein, the voltage can turned off, and the gel can be removed from the tube and separated by band.
Polyacrylamide gels are three-dimensional synthetic vinyl polymers most commonly formed from low molecular weight acrylamide and bisacrylamide monomers, the latter serving as a cross linker. Due to the action of an initiator, the monomer units in solution chemically bond to form a polymer with gel properties. However, the volume occupied by the polymerized gel is less than that of the monomer solution. Acrylamide shrinks about 0.25 milliliter (ml) per gram upon polymerization. Accordingly, this shrinkage can pull the gel away from the inner wall of the tube leaving non-sieving channels. These non-sieving channels diminish the ability of the gel column to separate molecular components during electrophoresis. Moreover, the gel can actually slide out of the tube during electrophoresis, impairing the separation process.
To alleviate the problem of non-sieving channels near the wall of the tube, the inner walls of capillary tubes have been coated with silylating or silanizing reagents, generally short, rigid carbon chains with a vinyl double bond at the free terminus. The acrylamide monomers bond covalently to the coating material as polymerization takes place so that the polymerized gel is tightly attached to the tube. This attachment minimizes the formation of non-sieving channels near the tube wall. However, the shrinkage induces stresses in the gel matrix so that voids can occur in the interior of the gel. These voids constitute gel inhomogeneities which disturb separation of sample components during electrophoresis and degrade gel column performance. Additionally, under the stress from gel shrinkage, pore sizes of the lattice network can be non-uniform.
U.S. Pat. No. 4,810,456 to Bente and Myerson discloses a technique to reduce gel shrinkage by applying high pressure to the monomer solution during polymerization. This method is applied in conjunction with coatings that bind the gel matrix to the column wall. The technique attempts to reduce shrinkage defects by compressing the monomer solution to a volume close to that expected of the polymerized gel, in other words, by preshrinking. However, this technique does not completely eliminate performance problems of the gel. The gels seem to be weaker near the center and do not last as long as desired. Additionally, the technique requires high pressure equipment which is costly.
What is needed is an electrophoretic gel column that can withstand greater electric fields and maintain its integrity for a greater number of runs. As a corollary, a method of making such a column is required, and the method should be economical and result in a gel which is securely attached to a column wall and free of interior voids.